NMR evidence for asymmetric radiation damage of bilayer liposomes

Radiat. Phys. Chem. Vol. 28, No. 4, pp. 355-358, 1986 Int. J. Radiat. Appl. lnstrum., Part C

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N M R E V I D E N C E FOR A S Y M M E T R I C R A D I A T I O N DAMAGE OF BILAYER LIPOSOMES H. SPRINZ, U. FRANCK, H. SCH~,FER and G. HOBNER Central Institute of Isotope and Radiation Research, Leipzig, 7050 Leipzig, Permoserstr. 15, G.D.R. (Received 21 March 1986)

Abstract--ln aqueous solutions of irradiated sonicated egg yolk lecithin vesicles the ~H relaxation times T~ and T~ were determined for the outer and inner (CH3)3N+-groups at 250 MHz and at room temperature. After a y irradiation up to a dose of 13.5 kGy, T 1 remains constant T I = (0.33 + 0.01)s, T2 is a sensitive parameter in detecting radiation induced changes of slow reorientations for the lipid molecules. While T2 = 0.10 s for the outer head group signal decreases by 25% after irradiation, the effect is significantly smaller for the inner head group. This preferred radiation damage of the outer lipid layer may be a consequence of the indirect radiation action and/or due to the geometric packing constraints in small vesicles. The spectroscopic results were derived from partially relaxed NMR spectra. This method seems to be useful for the detection of the effects of different agents on the radiation damage of the liposome, as demonstrated with the partially relaxed head group spectra in the presence of NaCI. (German abstract at end o f paper)

INTRODUCTION The observed radiation damage of lipids in aqueous solution is mainly due to the indirect radiation action mediated by the OH" radicals of the water radiolysis. tj':/ In single-liposome dispersions with a typical lecithin concentration of 5 w t % the fraction of the water inside the vesicles amounts to only a few percent of the total water. Therefore, one must suppose that more radicals interact with the outer layer than with the inner lipid layer and the question arises whether an asymmetry of the radiation action on the two lipid layers can be observed. Using the ianthanide shift method t31 we have shown by means of a careful lineshape analysis ~41 that the radiation induced line broadening of the I H - N M R signal of the (CH3)3H+-head group for the outer layer increases more rapidly with dose than for the inner layer. F r o m this result we have deduced a preferential radiation damage of the outer lipid layer. In this report we explore this asymmetry by proton relaxation methods using a high field N M R instrument with the advantage that a shift reagent as an additive is not necessary.

duced by ultrasonic treatment in an ice water bath (9 min, 20 kHz, 70 W) under an argon atmosphere. The liposomes were centrifuged (t> 60,000 g, 30 min). The resultant small unilamellar vesicles (5 wt% of lipid) were transferred into 5 m m N M R tubes and bubbled for 10min with nitrogen or air. The irradiation was performed in a GB 77 6°Co-irradiator (ZfI, Leipzig) with a dose rate of 3.4 Gy/min up to a dose of 13.5kGy at r o o m temperature. After irradiation the samples were bubbled for 10 rain with nitrogen. t H - N M R spectra were obtained on an A M 250 Fourier transform spectrometer (Bruker) at 21°C one day after irradiation. The spin-lattice relaxation time (T~) was measured using the inversion-recovery method. The spin-spin relaxation time (T2) was obtained by the Carr-Purcell/Meiboom-Gill sequence (CPMG). The time interval z between the first and the last pulse was longer than 0.02 s with both methods. The time dependence of the intensities of the partially related spectra follows a single exponential curve for all samples examined. ESR spectra were recorded on an E R 202 D LR spectrometer (Bruker) using a flat quartz cell with a thickness of 0.2 mm.

EXPERIMENTAL

Egg yolk lecithin was isolated according to Singleton et al. ~51DzO (99.92 a t % , Isocommerz G m b H ) was used as a solvent with NaC1 (1 mM) and without NaC1. Traces of DCI were added to provide a slightly acidic solution (pD ~ 5). The liposomes were p r o -

RESULTS AND DISCUSSION

With high field N M R the resonances of the outer and the inner head group signals of small liposomes can be partially resolved without shift reagents. 16~ 355

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#,

,sae

!55

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)z

1 3 5 k G y , N?

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Fig. I, Partially relaxed ~H-N M R spectra of the ((H,), N " group (Tr - z zr 2 sequence, -~ = 02 s) Using the preparation method described there results an area ratio of 1.9 : 1 for the outer to the inner head group signal) 4~ In the first series of experiments we used D,O with I m M NaCI as a solvent. For the outer head group of unirradiated and irradiated (air saturated) liposomes we measured Tx = (0.341 +_ 0.003) s and T~ = (0.326 +_ 0.010) s, respectively. Therefore we can conclude that the correlation time for the fast anisetropic rotational motions ~7) of that group is not influenced by irradiation up to this dose level A comparison of partially relaxed spectra (Fig. I ) shows that this statement also holds for the inner lipid layer. The relaxation process is somewhat faster than for the outer head group but independent of the dose. With the C P M G - m e t h o d T 2 = (0.102 ± 0.005) s and ~ = (0.077 ± 0.004) s were determined for the outer head group of unirradiated and irradiated (air saturated) liposomes, respectively. This decrease by 25% can be produced by paramagnetic influences, for example by radiation induced stable radicals at the outer surface of the liposomes. However, there is no detectable paramagnetic contribution to 7"~. Secondly, a comparison of the ESR spectra (20 scans) of an unirradiated and irradiated sample of an aqueous soluticn of liposomes made l h after irradiation (24 kGy, 400 Gy/min) shows the same small signals which are due to impurities in the quartz cell. which means that paramagnetic free radicals do not exist. A decrease of T, is expected if a radiation induced fusion of vesicles occurs. But our earlier N M R results have shown that immediately after irradiation the liposomes are still intact, as seen from the ratio of the areas of the outer to the inner head group signal which remains constant? 4j Furthermore, an irradiation experiment with a reduced lipid concentration shows that the decay of the outer signal is drastically accelerated (Fig. 2t. Consequently, the observed decrease of T: cannot be explained with a radiation induced aggregation of the liposomes, because the radiation effect is even higher at the lower liposome concentration in correspondance with the well known "dilution effect" in radiation chemistryP ~

Therefore, the shortening of 1": together with an unchanged T t points to the fact that the correlation times associated with slm~ reorientation processes relative to the axis normal la lhe bilayer surface increase, c~ The lateral diffusion o1" the lipid molecules and the slow segmental molions make the essenlial contributions to these reorieniation processes. '~'' Thc increase o f the rigidil} and the li~rnlation o1 crosslinks are discussed a~ radiation cltects on liposomes, c ' ' In fact. crosslinks max be ,,cry effective m reducing the translalional inoliol; The comparison o1" inlensitie> e l partially relaxed speclra was made with the aim (',1"determining small differences in the relaxation pn+cesse~ m the two lipid layers (Figs I and 2). Assuming a Lorentzian lineshape the ratio of the inten,qties ),1 ~+t the outc[ (o) to the inner (i) lipid laver can be expressed b> the equation I

]w: i~ i :

exp ,

r

t

i

T', •} 1

From this equation the ratio~ I : l . - 0 . 9 1 j_ t).04 and 1.07 + 0.03 were determined for the irradiated (air saturated) and unirradiated samples, respectively (Fig. 2).

. . . .

j~

#

J

\ \

iD "

, 3

~',

N

•

k

E

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Fig. 2. Transverse magnetization ol the ICH~ h N ' -protons after 100 ~-pulses for two different lipid concentrations ( C P M G - sequence, r - 0.2 s. A, B. C: 5 wt% lipid. 16 scans, D, E: 0.5 wt% lipid, 256 scans).

Radiation damage of bilayer liposomes

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protecting effect of NaCI (T°/T~ = 1.0) against irradiation.U i) The preferential radiation effect on T2 of the outer lipid layer may be caused by the geometric packing constraints in small vesicles. For instance, the acyl chain cross sections at the centre of the bilayer are equal to 0.46 nm 2 and 0.96 nm 2 for the outer and the inner acyl chain, respectively.(~2) Therefore, the formation of crosslinks may be favoured in the outer layer. A preferred radiation damage of the outer lipid layer should also be caused by the high fraction of OH" radicals produced in the outer bulk water. For the OH" radical, which is captured mainly by H2 and H202, we can estimate a lower limit of the life time equal to 10/as using G-values (number of molecules produced per 100 eV absorbed energy) for the H 2 and H202 formation. (s) Therefore, each OH" radical from the bulk water must be considered to be able to interact with the surface of the liposome at this lipid concentration. On the other hand, only a few percent of this amount of radicals can penetrate into the membrane from the internal water.

kGy

CONCLUSIONS

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02

The asymmetry found in the transverse magnetization decay for the outer and the inner head group signals can be explained with preferential radiation damage in the outer lipid layer at low salt concentrations and with a protecting effect of the Cl--ions(m at high salt concentrations. Partially relaxed NMR spectra offer a possibility to compare the relaxation process for the chemically identical head groups under identical experimental conditions. Acknowledgement--The authors are indebted to Dr S. Kuropteva for operating the ESR equipment at the Institute of Chemical Physics in Moscow.

I

I

3.30

I

I

3.20

I

I

I

3,10

8 (ppm}

Fig. 3. Transverse magnetization of the (CH3)3N ÷-protons after I00 n-pulses in dependence on the concentration of NaC1, added immediately before irradiation (CPMGsequence, z = 0.2 s, irradiation dose: 13.5kGy).

In a second set of experiments the partially relaxed CPMG-spectra were recorded for liposomes prepared in D20 without NaCI (Fig. 3), Different amounts of NaCI were added immediately before irradiation. These spectra clearly show the asymmetric radiation damage of the bilayer. In the solution without salt there is preferential radiation damage of the outer layer (T~/T~=0.86). In contrast, the spectrum for the solution with 0.15 M NaCI demonstrates the

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Zusammenfassung- An bestrahlten ultraschallbehandehen wfigrigen L6sungen ~on Eilecithin-Vesikeln wurden die Protonenrelaxationszeiten 7-~ und T, f~r die ~iugere und innere (CH~)~N ~-Kopfgruppe bei 250 M H z und bei Zimmertemperatur bestimmt. :Nach G a m m a b e s t r a h l u n g mit einer Dosis yon 13,5 kGy bleibt T~ konstant, T b - ( 0 , 3 3 t 0 , 0 1 ) s , w/ihrend T: einen empfindlichen Parameter zum Nachweis strahleninduzierter Ver/inderungen der langsamen Umorientierungen der Lipidmolekule darstellt. Wfihrend I'~ = 0.10 s nach Bestrahlung fiir die/iugere Kopfgruppe um 25% abnimmt, ist dieser Effekt f/Jr die innere l~opfgruppe deutlich kleiner. Diese bevorzugte Strahlensch/idigung der/iul3eren Lipidschicht kann eine Folge der indirekten Strahlenwirkung sein und/oder a u f die Geometrie der Lipidanordnung in kleinen Vesikeln zurOckzuffihren sere Die spektroskopischen Ergebnisse werden abgeleitet aus partiell relaxierten NMR-Spektren. Diese Methode scheint n/.itzlich zu sein zum Nachweis yon Einfl/issen verschiedener Stoffe a u f die Strahlensch/idigung yon Liposomen, wie an den partiell relaxierten Spektren der K o p ~ r u p p e n bei Anwesenheil von NaCI demonstriert wird.